Far from being a passive medium for sperm delivery, seminal fluid contains proteins that exert major influences on mated females. This is best understood in insects, where seminal fluid proteins ("Acps") stimulate ovulation and egg-laying, are required for a female to store sperm, and decrease her lifespan. Despite their importance, the molecular actions of seminal fluid proteins are unknown. We will use the tractable molecular genetics of Drosophila to determine how three major reproductive processes are controlled by Acps. In aim 1, we address how ovulation is stimulated by Acp26Aa, a male-derived prohormone. Using an Acp26Aa null mutation, we'll identify signals that transduce the effect of Acp26Aa. We will also identify the receptor through which Acp26Aa acts, and whether Acp26Aa needs processing, and its region similar to mollusk egg-laying hormones, to act. Our results are significant to reproduction, hormone action, and the evolution of reproductive isolation mechanisms. In aim 2, we investigate how seminal fluid proteins cause sperm storage. Again we'll work out from a single molecule, Acp36DE, that we showed genetically is central to this process. We will examine the behavior of GFP-labeled sperm in the absence of Acp36DE, testing whether Acp36DE acts within the sperm storage organs or to get sperm into storage. We will test whether Acp36DE's function requires its binding to sperm, and will identify molecules to which Acp36DE binds. Sperm storage is widespread among animals; our study will determine its molecular basis. In aim 3, we address the role of protease inhibitors in seminal fluid. Seminal fluid of many animals contains protease inhibitors, but their reproductive function is unknown. Acp62F is a trypsin inhibitor in Drosophila seminal fluid. Intriguingly, Acp62F is toxic when introduced into insect circulatory systems. We propose a mutational analysis to determine Acp62F's function in the seminal fluid, such as controlling proteolysis of crucial reproductive proteins. We will test if entry of Acp62F into the female's circulatory system during mating shortens her life. We will identify molecules which bind Acp62F; these should include the proteases it inhibits. Our results will establish reproductive roles for controlled proteolysis, and will test evolutionary hypotheses for why toxic proteins exist in semen. In aim 4, we'll continue screening a collection of EMS-induced mutations for ones in Acps. Analyzing these mutants, and ectopic expression of Acps, will identify new Acps that regulate ovulation, sperm storage or proteolysis.